Abstract
The catalytic inefficiencies of the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. Strategies to engineer more efficient plant Rubiscos have been hampered by evolutionary constraints, prompting interest in Rubisco isoforms from non-photosynthetic organisms. The methanogenic archaeon Methanococcoides burtonii contains a Rubisco isoform that functions to scavenge the ribulose-1,5-bisphosphate (RuBP) by-product of purine/pyrimidine metabolism. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 Å resolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L2)5, and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L8S8 enzymes. MbR contains a unique 29-amino acid insertion near the C terminus, which folds as a separate domain in the structure. This domain, which is visualized for the first time in this study, is located in a similar position to SSus in L8S8 enzymes between LSus of adjacent L2 dimers, where negatively charged residues coordinate around a Mg2+ ion in a fashion that suggests this domain may be important for the assembly process. The Rubisco assembly domain is thus an inbuilt SSu mimic that concentrates L2 dimers. MbR assembly is ligand-stimulated, and we show that only 6-carbon molecules with a particular stereochemistry at the C3 carbon can induce oligomerization. Based on MbR structure, subunit arrangement, sequence, phylogenetic distribution, and function, MbR and a subset of Rubiscos from the Methanosarcinales order are proposed to belong to a new Rubisco subgroup, named form IIIB.
Highlights
The catalytic inefficiencies of the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity
In form I Rubiscos, four L2 dimers are assembled around a 4-fold axis by two tetramers of ϳ15-kDa small subunits (SSu) that cap each end of the (L2)4 barrel
The Mg2ϩ ion is ligated by oxygen donor atoms provided by the side chains of Asp-366 and Asp-368 located at the N-terminal end of the Rubisco assembly domain (RAD) helix ␣J and Glu-179 and Asp-183 located in helix ␣1 of the ␣-barrel in the large subunits (LSu) of the neighboring L2 dimer (Fig. 1C and supplemental Fig. S1)
Summary
The catalytic inefficiencies of the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 Aresolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L2), and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L8S8 enzymes. In form I (higher plant, cyanobacterial, algal, and most proteobacterial) Rubiscos, four L2 dimers are assembled around a 4-fold axis by two tetramers of ϳ15-kDa small subunits (SSu) that cap each end of the (L2) barrel. These ϳ550kDa hexadecameric L8S8 Rubiscos exhibit far superior catalytic efficiency than their (L2)n counterparts. 4-CABP, 4-carboxyarabinitol 1,5-bisphosphate; XuBP, xylulose 1,5-bisphosphate; PDB, Protein Data Bank; r.m.s.d., root mean square deviation; E, expect value; RLP, Rubisco-like protein; MhR, M. hollandica Rubisco; H6-Ub, His6-ubiquitin; ESRF, European Synchrotron Research Facility; Bicine, N,N-bis(2-hydroxyethyl)glycine; IMAC, immobilized metal affinity chromatography
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